PROPOSAL SUMMARY Epidermal differentiation is critical for health with differentiation defects underlying skin diseases in children, including the common Atopic Dermatitis. Epidermal differentiation is a complex process in which enhancers, epigenetic changes, and transcription factors coordinately regulate the expression of structural proteins, adhesion molecules, and lipid producing enzymes necessary for epidermal barrier formation. Dramatic gene expression changes observed in differentiation correspond to large-scale reorganization of epigenetic regulator structures such as super-enhancers, which preferentially drive expression of genes vital for cell identity. In recent experiments, we have studied the dynamics of enhancer formation during differentiation of human keratinocytes. Within the super-enhancer encompassing the gene body of Grhl3, a key regulator of epidermal differentiation, we identified enriched binding sites for IRX, a transcription factor with unknown roles in the epidermis, suggesting that IRX may promote epidermal differentiation. Consistent with this idea, we found that the temporal expression of several IRX factors in mouse epidermal development mirrors that of known regulatory transcription factors for epidermal differentiation, including GRHL3. Depletion of GRHL3 in keratinocytes changes the enhancer landscape with formation of new non-keratinocyte super-enhancers that promote neuronal cell fate. Intriguingly, these newly formed enhancers exhibit striking overrepresentation of IRX binding sites, suggesting that GRHL3 may suppress the tendency of keratinocytes to exhibit neuronal-type features. The temporal expression pattern of several Irx genes in mouse epidermal development is similar to that of well-known epidermal differentiation. Single cell analysis of newborn mice epidermis unveiled a gradient of Irx3 expression that correlates with the keratinocyte's degree of differentiation. These preliminary results suggest that IRX plays a key role in epidermal differentiation. The hypothesis is that an interplay between GRHL3 and IRX promotes epidermal differentiation through at least two different mechanisms: 1) both IRX and GRHL3 activate genes expressing epidermal differentiation factors, and 2) GRHL3 suppresses the formation of IRX-sensitive SEs that otherwise would activate non-epidermal, neuronal genes in the differentiating epidermis. Two Aims are proposed to test the hypothesis: Aim 1 will utilize an in vitro model of human epidermal keratinocyte differentiation to provide insights into the mechanism whereby IRX regulates differentiation. Aim 2 will utilize conditional Irx3;Irx5 double knockout mice to characterize the epidermal differentiation phenotype of mice deficient for Irx3 and Irx5 in the epidermis. This work will establish the role of IRX in skin development and elucidate how GRHL3 and IRX collaborate in epidermal differentiation.